Massively parallel knock-in engineering of human T cells.

The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.

Mapping subcellular localizations of unannotated microproteins and alternative proteins with MicroID.

Proteogenomic identification of translated small open reading frames has revealed thousands of previously unannotated, largely uncharacterized microproteins, or polypeptides of less than 100 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and are often larger. The subcellular localizations of microproteins and alt-proteins are generally unknown but can have significant implications for their functions. Proximity biotinylation is an attractive approach to define the protein composition of subcellular compartments in cells and in animals. Here, we developed a high-throughput technology to map unannotated microproteins and alt-proteins to subcellular localizations by proximity biotinylation with TurboID (MicroID). More than 150 microproteins and alt-proteins are associated with subnuclear organelles. One alt-protein, alt-LAMA3, localizes to the nucleolus and functions in pre-rRNA transcription. We applied MicroID in a mouse model, validating expression of a conserved nuclear microprotein, and establishing MicroID for discovery of microproteins and alt-proteins in vivo.

Phosphorylation of a Human Microprotein Promotes Dissociation of Biomolecular Condensates.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation. One such intrinsically disordered microprotein is NBDY, a 68-amino acid component of membraneless organelles known as P-bodies. In this work, we show that NBDY can undergo liquid-liquid phase separation, a biophysical process thought to underlie the formation of membraneless organelles, in the presence of RNA in vitro. Phosphorylation of NBDY drives liquid phase remixing in vitro and macroscopic P-body dissociation in cells undergoing growth factor signaling and cell division. These results suggest that NBDY phosphorylation enables regulation of P-body dynamics during cell proliferation and, more broadly, that intrinsically disordered microproteins may contribute to liquid-liquid phase separation and remixing behavior to affect cellular processes.

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24.

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

Discovery of cellular substrates of human RNA-decapping enzyme DCP2 using a stapled bicyclic peptide inhibitor.

DCP2 is an RNA-decapping enzyme that controls the stability of human RNAs that encode factors functioning in transcription and the immune response. While >1,800 human DCP2 substrates have been identified, compensatory expression changes secondary to genetic ablation of DCP2 have complicated a complete mapping of its regulome. Cell-permeable, selective chemical inhibitors of DCP2 could provide a powerful tool to study DCP2 specificity. Here, we report phage display selection of CP21, a bicyclic peptide ligand to DCP2. CP21 has high affinity and selectivity for DCP2 and inhibits DCP2 decapping activity toward selected RNA substrates in human cells. CP21 increases formation of P-bodies, liquid condensates enriched in intermediates of RNA decay, in a manner that resembles the deletion or mutation of DCP2. We used CP21 to identify 76 previously unreported DCP2 substrates. This work demonstrates that DCP2 inhibition can complement genetic approaches to study RNA decay.

The NBDY Microprotein Regulates Cellular RNA Decapping.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in NBDY knockout (KO) cells reveals dysregulated stability of more than 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in NBDY KO cells, which correlates with 5' UTR length. NBDY deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay factors in NBDY KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.

Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines.

Ribosome profiling and mass spectrometry have revealed thousands of small and alternative open reading frames (sm/alt-ORFs) that are translated into polypeptides variously termed as microproteins and alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit stress-, cell-type-, and/or tissue-specific expression; understanding this regulated expression will be critical to elucidating their functions. While differential translation has been inferred by ribosome profiling, quantitative mass spectrometry-based proteomics is needed for direct comparison of microprotein and alt-protein expression between samples and conditions. However, while label-free quantitative proteomics has been applied to detect stress-dependent expression of bacterial microproteins, this approach has not yet been demonstrated for analysis of differential expression of unannotated ORFs in the more complex human proteome. Here, we present global micro-/alt-protein quantitation in two human leukemia cell lines, K562 and MOLT4. We identify 12 unannotated proteins that are differentially expressed in these cell lines. The expression of six micro/alt-proteins from cDNA was validated biochemically, and two were found to localize to the nucleus. Thus, we demonstrate that label-free comparative proteomics enables quantitation of micro-/alt-protein expression between human cell lines. We anticipate that this workflow will enable the discovery of regulated sm/alt-ORF products across many biological conditions in human cells.

MSN-on-a-Chip: Cell-Based Screenings Made Possible on a Small-Molecule Microarray of Native Natural Products.

Standard small-molecule microarrays (SMMs) are not well-suited for cell-based screening assays. Of the few attempts made thus far to render SMMs cell-compatible, all encountered major limitations. Here we report the first mesoporous silica nanoparticle (MSN)-on-a-chip platform capable of allowing high-throughput cell-based screening to be conducted on SMMs. By making use of a glass surface on which hundreds of MSNs, each encapsulated with a different native natural product, were immobilized in spatially defined manner, followed by on-chip mammalian cell growth and on-demand compound release, high-content screening was successfully carried out with readily available phenotypic detection methods. By combining this new MSN-on-a-chip system with small interfering RNA technology for the first time, we discovered that (+)-usniacin possesses synergistic inhibitory properties similar to those of olaparib (an FDA-approved drug) in BRCA1-knockdown cancer cells.

Protein-Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray.

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.

Accelerated cellular on- and off-target screening of bioactive compounds using microarrays.

In situ proteome labeling was carried out with 9 drug-like probes in live mammalian cells, with the corresponding cellular targets captured on microarrays and simultaneously screened using a diverse set of antibodies, revealing potential on- and off-targets.

Controlled proliferation and screening of mammalian cells on a hydrogel-functionalized small molecule microarray.

A hydrogel-functionalized small molecule microarray has been developed, on which PC-3 cancer cells were selectively grown. Subsequent controlled release of immobilized bioactive compounds enabled cell-based screening to be directly carried out on this platform.

A small-molecule protein-protein interaction inhibitor of PARP1 that targets its BRCT domain.

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high-throughput microplate-based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)-gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein-protein interactions. As the first known cell-permeable small-molecule PPI inhibitor of PAPR1, we further established that (-)-gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.

Microarray immobilization of biomolecules using a fast trans-cyclooctene (TCO)-tetrazine reaction.

By anchoring 1,2,4,5-tetrazine-containing biomolecules onto trans-cyclooctene (TCO)-functionalized slides, a site-specific microarray immobilization approach is described in this study. Compared with existing immobilization methods, our approach offers several distinctive features, including fast kinetics and high chemoselectivity.

Discovery of cell-permeable inhibitors that target the BRCT domain of BRCA1 protein by using a small-molecule microarray.

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50% of hereditary breast cancers. Cell-permeable, small-molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray-based platforms, we have discovered the first cell-permeable protein-protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)2 domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation-induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small-molecule inhibitor of poly-ADP-ribose polymerase) and Etoposide (a small-molecule inhibitor of topoisomerase II). Unlike previously reported peptide-based PPI inhibitors of BRCA1, our compounds are small-molecule-like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP-related pathways in DNA damage and repair response, and in cancer therapy.

"Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling.

Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.

Profiling human Src homology 2 (SH2) domain proteins and ligand discovery using a peptide-hybrid small molecule microarray.

A 396-member peptide-hybrid small molecule microarray was fabricated to profile small molecule ligands across 15 SH2 proteins, revealing hits against Lck and Grb2.

Site-specific immobilization of biomolecules by a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole.

We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.

Microarray-guided discovery of two-photon (2P) small molecule probes for live-cell imaging of cysteinyl cathepsin activities.

A microarray immobilized with 105 aldehyde-containing small molecules was screened against mammalian cell lysates over-expressing cathepsin L to identify two potent inhibitors, which were subsequently converted into cell-permeable probes capable of live-cell imaging of endogenous cysteinyl cathepsin activities by two-photon fluorescence microscopy.